Genome-wide profiling of Tbx6myc binding during zebrafish somitogenesis
Ontology highlight
ABSTRACT: Identification of Tbx6 genomic binding sites during zebrafish somitogenesis in order to identify Tbx6 targets genes Duplicate anti-myc ChIP samples with associated input for heat shocked Tg(hsp70l:tbx6Myc)v8 embryos, plus single paired anti-myc ChIP and input in wild type embryos - 3x ChIP samples; 3x input samples
Project description:Study of Smad2 and Eomesa genomic binding in zebrafish blastulas, their relationship to eachother and the relaptionship between genomic binding and Ndr1 and Eomesa responsive genes as identified by microarray and RNA-seq. Replicate ChIP samples with associated input for Smad2 and Eomesa - 4x ChIP samples (2 per factor); 2x input samples
Project description:The Myc-Max heterodimer has been thought of as a sequence specific DNA binding protein that regulates transcription of a large number of genes. We demonstrate here that the positions of the human genome occupied by Myc-Max correlate with the RNA polymerase II (Pol II) transcription complex rather than to the canonical DNA binding sequence element CACGTG. The heterodimer is positioned slightly upstream of essentially all promoter proximal paused polymerases and is found throughout the transcribed regions of genes. Using a multi-genome analysis of promoter regions we show that the heterodimers are oriented with respect to the direction of transcription with Myc downstream of Max. In strong support of a model in which the Myc is recruited by transcription complexes rather than specific DNA sequences, we found that the difference in affinities of Myc-Max heterodimers for CACGTG versus non-specific DNA is not great enough to drive the pattern of genome occupancy exhibited. A total of 2 ChIP-Seq data for Myc and Max in human HeLa cell.
Project description:Contemporary high throughput technologies permit the rapid identification of transcription factor (TF) target genes on a genome-wide scale, yet the functional significance of TFs requires knowledge of target gene expression patterns, cooperating TFs and cis-regulatory element (CRE) structures. Here we investigated the myogenic regulatory network downstream of the Drosophila zinc finger TF Lame duck (Lmd) by combining both previously published and newly performed genomic data sets, including chromatin immunoprecipitation sequencing (ChIP-seq), genome-wide mRNA profiling, cell-specific expression patterns of putative transcriptional targets, analysis of histone mark signatures, studies of TF co-occupancy by additional mesodermal regulators, TF binding site determination using protein binding microarrays (PBMs), and machine learning of candidate CRE motif compositions. Our findings suggest that Lmd orchestrates an extensive myogenic regulatory network, a conclusion supported by the identification of Lmd-dependent genes, histone signatures of Lmd-bound genomic regions, and the relationship of these features to cell-specific gene expression patterns. The heterogeneous co-occupancy of Lmd-bound regions with additional mesodermal regulators revealed that different transcriptional inputs are used to mediate similar myogenic gene expression patterns. Machine learning further demonstrated diverse combinatorial motif patterns within tissue-specific Lmd-bound regions. PBM analysis established the complete spectrum of Lmd DNA binding specificities, and site-directed mutagenesis of Lmd and additional, newly discovered motifs in known enhancers demonstrated the critical role of these TF binding sites in supporting full enhancer activity. Collectively, these findings provide new insights into the transcriptional codes regulating muscle gene expression, and offer a generalizable approach for similar studies in other systems. Examination of Lmd occupancy to genomic DNA from sorted mesodermal cells
Project description:Elucidating the role of gut microbiota in physiological and pathological processes has recently emerged as a key research aim in life sciences. In this respect, metaproteomics (the study of the whole protein complement of a microbial community) can provide a unique contribution by revealing which functions are actually being expressed by specific microbial taxa. However, its wide application to gut microbiota research has been hindered by challenges in data analysis, especially related to the choice of the proper sequence databases for protein identification. Here we present a systematic investigation of variables concerning database construction and annotation, and evaluate their impact on human and mouse gut metaproteomic results. We found that both publicly available and experimental metagenomic databases lead to the identification of unique peptide assortments, suggesting parallel database searches as a mean to gain more complete information. Taxonomic and functional results were revealed to be strongly database-dependent, especially when dealing with mouse samples. As a striking example, in mouse the Firmicutes/Bacteroidetes ratio varied up to 10-fold depending on the database used. Finally, we provide recommendations regarding metagenomic sequence processing aimed at maximizing gut metaproteome characterization, and contribute to identify an optimized pipeline for metaproteomic data analysis.
Project description:We performed ChIP-Seq analysis of SOX10, histone H3 lysine 27 acetylation (H3K27ac) and H3K27 trimethylation (H3K27me3) in melanocytes to profile the genomic binding sites of SOX10 and the chromatin landscape. In parallel, we generated Sox10 haploinsufficient cell lines using gene knockout approaches and conducted microarray gene expression analysis to identify functional gene targets of SOX10 transcriptional regulation in melanocytes. We demonstrate that SOX10 predominantly engages “open” chromatin, binds to melanocyte enhancer elements and plays a central role in transcriptional activation and repression of functionally distinct classes of genes. Furthermore, we identified cis-regulatory sequence motifs of putative co-regulatory transcription factors that define SOX10-activated and SOX10-repressed target genes. Our results uncover novel mechanisms and roles of SOX10 in global transcriptional regulation of diverse regulatory pathways in the melanocyte lineage. ChIP-seq profiling of SOX10, H3K27ac, and H3K27me3 in the mouse melanocyte cell line melan-Ink4a-Arf-1 (melan-a).
Project description:The tumor suppressor p53 has been studied extensively as a direct transcriptional activator of protein-coding genes. Recent studies, however, have shed light on novel regulatory functions of p53 within noncoding regions of the genome. Here, we use a systematic approach that integrates transcriptome-wide differential expression analysis, genome-wide p53 binding profiles, chromatin state maps, and additional genomic features to characterize the global regulatory roles of p53 in response to DNA damage in both human and mouse fibroblast models. In addition to known p53 targets, we identify many previously unappreciated mRNAs and long noncoding RNAs that are regulated by p53. Moreover, we find that p53 binding events occur predominantly within enhancer elements in both human and mouse systems. The ability to modulate enhancer activity offers an additional layer of complexity to the p53 network and greatly expands the diversity of genomic elements that are directly regulated by p53. Human and Mouse fibroblasts cultured in the presence or absence of doxorubicin followed by RNA-Seq (Human:2 cell lines, each condition in duplicate; Mouse:MEF cell line,each condition in triplicate) and p53 ChIP-Seq (Human:2 cell lines, input and IP for each; Mouse:MEF cell line, input and IP)
Project description:The Mammalian cardiomyocyte (CM) cell cycle is tightly regulated by a complex network of transcription factors; however, the gene regulatory networks depending on their developmental stages are unclear. Here, we report that Tbx6, expressed in the cardiac mesoderm, promote cell cycle re-entry in vitro and in vivo. We performed a gain of function screening for cardiac development regulator genes that promoted neonatal CM proliferation. Tbx6 activate the cell cycle and promote cultured neonatal CM proliferation. Next, we generated recombinant adeno-associated virus serotype 9 vector encoding Tbx6 (AAV9-Tbx6). Injection of AAV9-Tbx6 was shown to promote cell cycle re-entry of CMs in both postnatal and adult mice. Tbx6 overexpression activated cell cycle activators, while repressed cell cycle inhibitor gene. Thus, Tbx6 promotes neonatal CM proliferation in vitro and adult CM cell cycle re-entry in vivo.
Project description:Msgn1 is a bHLH transcription factor and is a direct target gene of the Wnt/b-catenin signaling pathway. During mouse embryogenesis, Msgn1 is expressed in the mesodermal compartment of the primitive streak and is required for the differentiation of presomitic mesoderm. Msgn1-/- mutants show defects in somitogenesis leading to a lack of trunk skeletal muscles, vertebra and ribs. The goal of this study is to dissect the molecular and cellular function of Msgn1 in Embryonic Stem Cells (ESC) and mouse development. In order to identify direct Msgn1 targets, we performed transcriptional profiling and CHIP-seq of Msgn1 expressing differentiating ES cells. Integration of these data sets, we found that Msgn1 is a master regulator of PSM differentiation regulating gene expression programs of PSM identity, EMT, motility and Notch segmentation clock. Inducible Flag Msgn1 ES cells were differentiated to form Embryoid bodies (EBs) for 2 days. Flag-Msgn1 was induced on day 2 with doxycycline and samples were collected 36h later. Here Input DNA is used as control.